Journal
BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION
Volume 32, Issue 4, Pages 228-235Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1002/bmb.2004.494032040358
Keywords
enzyme kinetics; slow-binding inhibition; linear and nonlinear regression; stopped flow; progress curves
Ask authors/readers for more resources
Tyrosinase (EC 1.14.18.1) catalyzes the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) to 2,3,5,6-tetrahydro-5,6-dioxo-1H-indole-2-carboxylate (dopachrome), according to the classical Michaelis-Menten kinetic mechanism. The enzyme is strongly but slowly inhibited by alpha-amino-beta-[N-(3-hydroxy-4-pyridone)] propionic acid (L-mimosine), a toxic plant amino acid. Easily available reagents and simple spectrophotometric detection of the product make the experimental characterization and kinetic analysis of tyrosinase action convenient and interesting for teaching purposes. In the present article, we present a theoretical and practical guide to the kinetic analysis of slow-binding inhibition. The effect Of L-mimosine on tyrosinase is established by progress curve measurements, carried out on a conventional spectrophotometer equipped with a rapid kinetic accessory. In the analysis, we recommend a classical linearization approach but we also took advantage of a more reliable nonlinear regression method to avoid subjective bias. A multistep procedure starts by careful inspection of the curves to discriminate between candidate mechanisms. Next, the evaluation of initial and steady-state velocities provides information on the enzyme catalytic and Michaelis-Menten constants, as well as the corresponding inhibition constants. Subsequently, an appropriate mathematical derivation enables estimation of the isomerization rate constants characteristic for a slow-binding inhibitor. To conclude, we suggest simultaneous multivariable regression, using all the progress curve data, to cross-check the proposed reaction mechanism and evaluated kinetic constants.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available